The genome sequence of the Oak Nycteoline moth, Nycteola revayana (Scopoli, 1772)

We present a genome assembly from an individual male Nycteola revayana (the Oak Nycteoline moth; Arthropoda; Insecta; Lepidoptera; Nolidae). The genome sequence is 621.0 megabases in span. Most of the assembly is scaffolded into 26 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.25 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,235 protein-coding genes.


Background
The Oak Nycteoline Nycteola revayana is a small moth in the family Nolidae, superfamily Noctuoidea, found widely across western, central and northern Europe, with scattered records from Russia and north Africa (GBIF Secretariat, 2023).In Britain and Ireland, the moth is recorded most frequently in southern and central England and Wales but rarely in large numbers; it has a scattered distribution across Scotland and is scarce in Northern Ireland and Ireland (GBIF Secretariat, 2023;NBN Atlas Partnership, 2023;Randle et al., 2019).An unusual feature of this species is the extensive variation in ground colour of the wings and in wing pattern.For example, moths may be ashy grey with indistinct markings, bronze-brown with pronounced black streaks running from the wing base, pale grey with 'domino-like' spots, or crossed by bands of brown, black and grey.In 1919, W.G. Sheldon combined previous work with examination of additional specimens to divide the species into 30 named 'varieties' (Sheldon, 1919).However, since intermediate forms exist, these may be better considered simply as combinations of variants in several independent wing pattern elements, rather than distinct morphs with co-inherited features.
The larvae of N. revayana feed on pedunculate oak Quercus robur and sessile oak Q. petraea, and the moth is found in deciduous woodlands or in fields, parks and hedgerows where oaks are present.In Britain, the species is thought to be primarily bivoltine: one generation is on the wing in autumn and spring, overwintering as an adult, with the eggs laid by these moths developing into a summer generation with more individuals.However, this pattern may be overlain by some moths having a univoltine life history, and supplemented by migrant moths from mainland Europe (DorsetMoths, 2023;Randle et al., 2019).
A genome sequence for N. revayana will facilitate studies into adaptations to oak feeding and the genetic basis of wing pattern variation, and will contribute to the growing set of genomic resources for Lepidoptera.

Genome sequence report
The genome was sequenced from a male Nycteola revayana (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34).A total of 33-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 8 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the scaffold number by 4.26%.
The final assembly has a total length of 621.0 Mb in 44 sequence scaffolds with a scaffold N50 of 22.0 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.79%) of the assembly sequence was assigned to 26 chromosomallevel scaffolds, representing 24 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
The estimated Quality Value (QV) of the final assembly is 66.3 with k-mer completeness of 100.0%, and the assembly has a BUSCO v completeness of 98.4% (single = 98.2%, duplicated = 0.2%), using the lepidoptera_odb10 reference set (n = 5,286).

Sample acquisition and nucleic acid extraction
Two Nycteola revayana (ilNycReva1 and ilNycReva2) were collected from Wytham Woods, Oxfordshire (biological  vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 2021-03-31 by Douglas Boyes (University of Oxford), using a light trap.The specimens were identified by the collector and then snap-frozen on dry ice.Individual ilNycReva1 (specimen Ox001089) was used for genome sequencing, while ilNycReva2 (specimen Ox001090) was used for Hi-C scaffolding.
The specimen used for RNA sequencing (specimen ID NHMUK010636607, ToLID ilNycReva3) was collected

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences Sequel IIe (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from $HIC_TISSUE tissue of ilNycReva2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly and curation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021).Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and PretextView (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Final assembly evaluation
The final assembly was post-processed and evaluated with the three Nextflow (Di Tommaso et al The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline (Challis et al., 2020).It aligns the PacBio reads with SAMtools and mini-map2 (Li, 2018) and generates coverage tracks for regions of fixed size.In parallel, it queries the GoaT database (Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO (Manni et al., 2021).For the three domain-level BUSCO lineage, the pipeline aligns the BUSCO genes to the Uniprot Reference Proteomes database (Bateman et al., 2023) with DIAMOND (Buchfink et al., 2021) blastp.The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx.Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn (Altschul et al., 1990).All those outputs are combined with the blobtools suite into a blobdir for visualisation.
All three pipelines were developed using the nf-core tooling (Ewels et al., 2020)

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Nycteola revayana assembly (GCA_947037095.2) in Ensembl Rapid Release at the EBI.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material Authors provide a sufficient introduction and rationale behind sequencing this genome, and I would not consider that there is anything major (or minor) left behind.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Evolutionary biology of butterflies including phylogenetics and biogeography.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

I only have two points that need further clarification:
In the Background "... into adaptations to oak feeding" is this remarkable for this species?If so, more should be given as to why this is important. 1.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Bacterial genomics, arthropod genomics, viral genomics, vector-borne disease, molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 11 September 2024 https://doi.org/10.21956/wellcomeopenres.23839.r92998I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Nycteola revayana, ilNycReva1.2:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 621,039,493 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (41,453,523 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,960,024 and 18,563,743 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Nycteola_revayana/dataset/GCA_947037095.2/snail.

Figure 4 .
Figure 4. Genome assembly of Nycteola revayana, ilNycReva1.2:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Nycteola_revayana/dataset/GCA_947037095.2/cumulative.

Figure 5 .
Figure 5. Genome assembly of Nycteola revayana, ilNycReva1.2:Hi-C contact map of the ilNycReva1.2assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=EP63k2ELTVC8tQ8OwXI0mg.

©
2024 Menezes R.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Rodolpho S.T MenezesDepartment of Biological Sciences, State University of Santa Cruz, Ilhéus, BrazilThe data note by Boyes et al. presents a genome assembly from an individual male of the moth Nycteola revayana (Scopoli, 1772).The final assembly is 621 Mb.This genome information will be interesting for comparative genomics investigation and also for evolution and conservation studies.The methodology is thorough and explained and the figures are well constructed, and the data note is well written.I have no notes to add.Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Cytogenetics, Phylogenomics, and Phylogeography.